Despite the availability of drugs and a partially-efficacious vaccine, tuberculosis remains an enormous health problem worldwide, due in part to the ability of M. tuberculosis to evade innate and adaptive immune responses. Development of economically-feasible improved methods of control of tuberculosis requires better understanding of the mechanisms underlying the development of immune responses to M. tuberculosis, and better understanding of the limitations of those responses. In the previous project period, we developed and used several novel tools and techniques in a mouse model to discover that M. tuberculosis-specific CD4+ T cell responses are initiated 10-11 days after infection of the lungs, occur earliest in the mediastinal lymph node, depend on transport of M. tuberculosis from the lungs to the lymph node by myeloid dendritic cells (DCs), and depend on production of antigen by bacteria in the lymph node, not the lungs. In addition, we determined that, while myeloid DCs transport M. tuberculosis to the lymph node, they are not the most efficacious antigen- presenting cells in the lymph node of M. tuberculosis-infected mice. These results indicate that better understanding of the immune response to M. tuberculosis and its limitations depends on better understanding of the events that precede and the events that follow acquisition of M. tuberculosis by DCs in the lungs. To better understand the mechanisms that precede acquisition of M. tuberculosis by DCs in the lungs, we will test the hypotheses that DCs do not acquire M. tuberculosis until 7-10 days after infection of the lungs, that interactions with lung epithelial cells and with neutrophils modulate acquisition of M. tuberculosis by DCs, and that bacterial inhibition of apoptosis contributes to delayed acquisition of M. tuberculosis by DCs in the lungs. To better understand the mechanisms of M. tuberculosis transport from the lungs to the mediastinal lymph node for initiation of the adaptive immune response, we will test the hypothesis that the chemokine receptors CCR7 and CCR8 mediate migration of mature DCs from the lungs to the mediastinal lymph node. We will also determine the respective roles of lymph node myeloid, lymphoid, and plasmacytoid DCs in initiating the adaptive immune response to M. tuberculosis. We will determine whether lymphoid and/or plasmacytoid DCs can present M. tuberculosis antigens after myeloid DCs transport the bacteria from the lungs to the lymph node, we will determine whether populations of lymphoid and plasmacytoid DCs acquire and present a secreted M. tuberculosis antigen in the lymph node during infection, and we will determine the respective contributions of individual infected and uninfected cells in presenting a mycobacterial peptide antigen in vivo. The proposed studies will yield unique new information on the events and mechanisms that contribute to initiation of adaptive immunity to M. tuberculosis, and will reveal specific targets for modulation to accelerate development of the adaptive immune response, to make it more efficacious. PUBLIC HEALTH RELEVANCE The immune response to Mycobacterium tuberculosis is only partially effective, as it allows survival of the bacteria and reactivation of disease in a large number of people. We have used a mouse model of tuberculosis to understand how the immune response is initiated during infection, and found that specific cells must carry the bacteria from the lungs to a local immune organ, called the mediastinal lymph node. In this project, we will determine how those specific cells acquire the bacteria, transport them from the lungs to the mediastinal lymph node, and stimulate lymphocytes to control the infection, to guide development of better ways to get the immune system to control tuberculosis.